Synergetic effect of essential oils and calcium phosphate nanoparticles for enhancement the corrosion resistance of titanium dental implant

Calcium phosphate (CaPO4) coating is one of various methods that is used to modify the topography and the chemistry of Ti dental implant surface to solve sever oral problems that result from diseases, accidents, or even caries due to its biocompatibility. In this work, anodized (Ti-bare) was coated by CaPO4 prepared from amorphous calcium phosphate nanoparticles (ACP-NPs) and confirmed the structure by X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR) techniques. Ti-bare was coated by prepared CaPO4 through the casting process, and the morphology of Ti/CaPO4 was characterized by scanning electron microscope (SEM) where the nano-flakes shape of CaPO4 and measured to be 60 ~ 80 nm was confirmed. The stability of Ti-bare and coated Ti/CaPO4 was studied in a simulated saliva solution using electrochemical impedance spectroscopy (EIS) and linear polarization techniques to deduce their corrosion resistance. Furthermore, three essential oils (EO), Cumin, Thyme, and Coriander, were used to stimulate their synergistic effect with the CaPO4 coat to enhance the corrosion resistance of Ti implant in an oral environment. The fitting EIS parameters based on Rs [RctC]W circuit proved that the charge transfer resistance (Rct) of Ti/CaPO4 increased by 264.4, 88.2, and 437.5% for Cumin, Thyme, and Coriander, respectively, at 2% concentration.


Experimental work Anodization
Through the anodization process, the layer of TiO 2 was formed as follows: the Ti-foil with an area of 1 cm × 1 cm and thickness of 1mm (99.99% metal basis, Sigma Aldrich) was used as the starting material.First, the Ti degreasing was performed by sonication in a mixture of acetone, isopropanol, and methanol.Then, the foil was washed with deionized water and dried in nitrogen steam.The Ti sample was connected at the positive pole of the power supply and immersed in a solution of 1.0 M H 3 PO 4 + 0.8 wt.% NaF doing as oxidants in the electrochemical cell at a constant potential of 20 V for 30 min.Finally, the sample after anodization was cleaned by immersion several times in deionized water and dried overnight in an oven under a nitrogen atmosphere, and then the Ti-bare sample was ready.The main goals for surface treatment through anodization are enhancement of bioactivity, biocompatibility, and corrosion resistance for dental applications.

Preparation of ACP-NPs
Potassium hydrogen phosphate was dissolved in deionized water at room temperature, along with the addition of calcium nitrate to form a P-precursor Ca-precursor solution with concentrations 0.040 M and 0.036 M, respectively.The pH value of the solution was fixed and adjusted at 8.0 by appending ammonium hydroxide solution (0.1 M) while continuously stirring at room temperature (25 °C) until a white slurry formation was achieved.The solution was centrifuged at 7000 rpm for 10 min and washed with distilled water three times.The prepared calcium phosphate precipitate was washed with acetone three times and dried at room temperature for 24 h; they were later stored under a vacuum for chemical structure characterization by XRD & FT-IR and further electrochemical studies.
www.nature.com/scientificreports/Electrochemical measurements of prepared Ti/CaPO 4   To produce a new modified Ti implant surface by ACP-NPs and subsequently evaluate its electrochemical behavior, a working electrode was utilized in the experiment.It consisted of an anodized sample Ti-bear with a surface area of 0.0625 cm 2 and a thickness of 1 mm.Initially, the surface underwent a polishing process utilizing a gentle emery paper, followed by a thorough rinse with double distilled water and ethanol.
The ACP-NPs were prepared from 50 mg calcium triphosphate and suspended in 1 mL of ethanol.The 100 µL of suspension was cast on the anodized Ti-bare surface.Finally, the surface was dried overnight.The samples were nominated by Ti/CaPO 4 when not exposed to the essential oils, but when exposed to EO named based on the type of the oil used as Ti/CaPO 4 -L1, Ti/CaPO 4 -L2, and Ti/CaPO 4 -L3 for Cumin, Thyme, and Coriander oils, respectively.The essential oils were supplied by local brand "Harraz" the concentration of pure essential oil is ~ 100%.
EIS and polarization results were achieved using Autolab PGSTAT128N.The electrochemistry software NOVA fitted the impedance spectrum (Version 2.1, Metrohm Autolab, Utrecht, Netherlands).A three-electrode cell was used, utilizing distinct surfaces as working electrodes.The reference and auxiliary electrodes employed were Ag/ AgCl/KCl (saturated) and Pt wire, respectively.
The artificial saliva was prepared as mentioned in Table 1.That the pH was adjusted to 7.4.

Surface characterization
The chemical structure of both samples, anodized titanium Ti-bare and coated Ti/CaPO 4 , were characterized by X-ray diffraction techniques.As represented in Fig. 1a, the XRD chart for anodized Ti surface..
The bond stretching within the molecule of the prepared CaPO 4 was studied by the FT-IR spectroscopy method to determine the functional groups.As illustrated in Fig. 2a.The peaks in the range of 3427 cm −1 and 1639.1 cm −1 refer to O-H stretching vibration modes of adsorbed water traces 65 .In addition, the peaks at 1034 and 563.3 cm −1 correspond to the orthophosphate group vibration modes 66 .Furthermore, the IR spectra for different essential oils were studied as represented in Fig. 2b. the peak observed at 3446 cm -1 corresponding to the O-H stretching in water molecule on the surface 44 .else, the peak at wavenumber equal to 2994, and 2820, and 1749 cm −1 attributed to the functional groups of (sp3)C-H, and -(O=C-H) and C=O respectively [67][68][69] .The surface morphology of the CaPO 4 , which will deposit on the anodized Ti surface, was characterized by SEM, as represented in Fig. 3, at two different magnifications.As represented in Fig. 3, the nanosized CaPO 4 particles were confirmed, and it was found to have a nano-flake shape and measured to be 60 ~ 80 nm.This figure illustrates more cavities on the surface, which increases the surface roughness, which is important to raise the biocompatibility of the Ti surface in saliva solution via increasing the corrosion resistance, as illustrated in the following sections.As we know, ACP is essential for the formation of mineralized bone and is used for bone substitutes 70 .
In the following sections, the electrochemical behavior of the anodized Ti/CaPO 4 samples in compared with Ti-bare metal was investigated through the electrochemical impedance studies to estimate the corrosion resistance of these samples towards the simulated physiological solutions which containing the concentrations of salts and ions like; Cl, Na, P that play a responsible role in changing on the stability of these surfaces.We will discuss in detail the interpretation of the EIS measurements for the coated Ti/CaPO 4 sample with or without the effect of essential oils (EO) in comparison with the Ti-bare sample.During the fitting process, the EIS data were fitted using ANOVA software, and the fitted circuit is illustrated in Fig. 4. The fitting model consists of solution resistance (R s ) connected in series with (R c //C), where R c is a charge transfer resistance, and C is a capacitor element.The presence of a constant phase element regards the nationally formed anatase layer (TiO 2 ), as reported in the XRD part.Furthermore, the constant phase element of EIS is mathematically like the capacitor component.Where the following equation can employ the impedance of CPE 71-73 : where the proportional factor (Y o ) is the CPE constant, the angular frequency is (ω) (in radians/sec), the imaginary number j 2 = − 1, and n is the CPE exponent ranges from zero to one.
Given the EIS Nyquist spectra of the present samples after 3 h of immersion in simulated saliva solution containing EO, was represented in Fig. 5.We noticed that the EO (L1 or L2 or L3) plays a great role in raising the corrosion resistance of the anodized Ti/CaPO 4 .These results support our hypothesis that while the CaPO 4 on anodized Ti acts as a bioactive coat, a synergistic effect emerges between the EO CaPO 4 , acting as a superior for the stability of Ti sample in the oral environment.In the following sections, we will be giving more evidence  Figure 6a,b shows the Nyquist plot of the Ti-bare and Ti/CaPO 4 surface through 240min.As represented in Fig. 6a, the Nyquist plot of Ti-bare in saliva solution shows the increase of resistance by time of soaking.As appeared in Fig. 6b, the value of the impedance increases in the resistive component, which indicates that the CaPO 4 coat enhances the corrosion resistance by improving the surface compatibility 74 .
A comparison between the charge transfer resistance, calculated over different time intervals for Ti-bare and Ti/CaPO 4 electrodes, was represented in (Fig. 6c).Where the R ct values for Ti-bare are less than Ti/CaPO 4 values, this explains the role of CaPO 4 in enhancing the corrosion resistance between the coating and Ti surface in saliva solution.Additionally, the fitting parameters of Ti-bare and modified Ti/CaPO 4 by using the fitting circuit appeared in Fig. 4, are represented in Tables 2 and 3. From this Table, we can deduce the many important features as the resistance of Ti-bare redouble from 424 Ω to 844 Ω after 240 min (Table 2), while for Ti/CaPO 4 it 4 times multiplied during the same time, where the resistance increases from 460.08 to 1875.3 Ω (Table 3).Also, the high-value R s values of Ti-bare than Ti/CaPO 4 samples refers to the role of CaPO 4 to decrease the R s values during the time of immersion, which means that the good migration of the ions to the surface.The small capacitance value C points to the low capability of the surface to store charge.This result supports the good corrosion resistance of the surfaces where the capacitance is inversely proportional to the resistance 70 .
The effect of increasing the time of immersion from 240 min to 336 h or 14 days was studied in this section.As seen in Fig. 7a,b, the Nyquist plot of the Ti-bare and Ti/CaPO 4 after 14 days of soaking in the saliva solution (without EO).The linear Nyquist plot indicates a non-charge transfer process, whereas the process is mainly diffusion 70 .Another time, the increase of the corrosion resistance of the Ti/CaPO 4 sample reflects the positive impact of the CaPO 4 layer, where the coating layer promotes surface durability.In Fig. 7c, a comparison between the charge transfer resistance over different time intervals for Ti-bare and Ti/CaPO 4 electrodes was studied, and the results indicated that the charge transfer resistance reached a steady state after 14 days of soaking in the saliva solution.The resulting EIS fitting parameters based on the same fitting circuit (Fig. 4) were reported in the Tables 4 and 5.Where the resistance increases from 2831 to 3985 Ω for Ti-bare and from 4624 to 6890 Ω for Ti/CaPO 4 during 336 h.www.nature.com/scientificreports/In this section, and for further enhancement of the corrosion resistance of the coated Ti/CaPO 4 surfaces.Three EO were used for medical applications, named Cumin, Thyme, and Coriander oils (L1, L2, and L3).They injected into the operating solution.The effect of EO was studied over two periods, 240 min and 336 h, to compare the result of the same electrodes in the presence of the green extract.As represented in Fig. 8, the comparison between the effect of concentration of three EO on the charge transfer resistance Rct of Ti/CaPO 4 appeared.Whereas different oil concentrations were used, i.e., 0.25% up to 2% (Wt/Wt).Rct of Ti/CaPO 4 was observed to increase by 264.4,88.2, and 437.5% for L1, L2, and L3, respectively, at 2% of EO concentration.
As illustrated in Fig. 9a-c, the Nyquist plot of modified Ti/CaPO 4 electrode in saliva solution in the presence of 2.0% of different essential oils (L1, L2, and L3) for different time intervals up to 4 h. of soaking.However, the oil started diffusion with time, and the oil adsorption on the electrode surface enhanced the corrosion resistance over time.Hence, the diffusion of the saliva solution to the inner layers of the Ti sheet promotes the layer of the anodized Ti form, which is important for the corrosion resistance process.The progress of charge transfer resistance for each oil was followed as represented in Fig. 9d.By fitting the EIS result based on the fitting circuit (Fig. 4) for the Nyquist data, the Rct of the electrodes increased by 85, 27, and 33% for L1, L2, and L3, respectively.www.nature.com/scientificreports/ The change in the charge transfer value reflects the different abilities of an oil to be adsorbed on the Ti surface.
Whereas the oil ingredients are different in chemical structures.The fitting parameters extracted from Nyquist plots are represented in Tables 6, 7 and 8.The modified electrode Ti/CaPO 4 was investigated in saliva solution and injected oils for 14 days (see Fig. 10a,c).After the 336 h of soaking of Ti/CaPO 4 in saliva, the electrode resistance started to reach the steady state where the charge transfer resistance was slightly changed.In the last 7 days, the resistance changed by 1.9, 7, and 2.8% for L1, L2, and L3, respectively.The anticorrosion activity of the EO was in the order of L3 > L1 > L2.The steric hindrance of the active component in the essential oils plays an important role in corrosion inhibition.For L3 oil, the most common ingredient, linalool, is long-chain alcohol, which has a smaller molecular volume than the bulky Thymol group (L2).The bulky group around the oxygen atom in thymol decreases the ability of thymol to be adsorbed on the titanium surface and diffusion through the CaPO 4 layer.As shown in Tables 9, 10 and 11, the EIS-relevant data were reported for different essential oils corresponding to the different time intervals.The progress of charge transfer resistance for each oil was shown in Fig. 10d, where the promising effect of L3 & L1 appeared and reached the high value after 72 h to be stable at 336 h Although the value of the corrosion resistance is nearly the same for both L3 & L1 at the beginning of soaking (8166.5 & 8405 Ω, respectively), it doubles more than twice in the case of the L3 (20421Ω), compared to one and half times in the case of L1 (14,523 Ω) after 336 h at the ending soaking.While the L2 oil increases the corrosion resistance with low value in comparison to L1& L3, the progress of the resistance tripled through the time of soaking from 2680 to 7525 Ω.

Polarization technique
The Tafel technique was used to prove the best behavior of surfaces in saliva solution after 14 days of immersion.As represented in Fig. 11, the linear polarization curve of different modified electrodes Ti-bare, Ti/CaPO 4 , Ti/ CaPO 4 -L1, Ti/CaPO 4 -L2, and Ti/CaPO 4 -L3 at a scan of 1 mV s −1 within potential window − 1 to 1 V vs. the open circuit potential.The linear polarization data can estimate the Tafel slopes (βa and βc).Furthermore, the values of corrosion current density (I corr ) and corrosion potential (E corr ) can be calculated by the interception of Tafel lines as listed in Table 12.The coated Ti/CaPO 4 showed more positive corrosion current (E corr ) values than the Ti-bare electrode, indicating that lower corrosion rate, which is less favored upon these surfaces.On the other hand, the corrosion current is a value that reflects the actual corrosion rate.The electrode with a higher corrosion   www.nature.com/scientificreports/current indicates a higher corrosion rate.The effect of essential oil addition was observed for the following modified surfaces: Ti/CaPO 4 -L1, Ti/CaPO 4 -L2, and Ti/CaPO 4 -L3.Whereas the E corr of the modified electrode showed an order of L3 > L2 > L1, this result is equivalent to the result deduced from the EIS results.Furthermore, the polarization resistance reflects the current flow rate caused by electrochemical reactions.The sample with higher polarization resistance matched the sample with higher corrosion resistance.Also, the corrosion rates of   each electrode were estimated using NOVA software based on the polarization data (See Table 12).The corrosion rate was expressed as mm of Ti foil per year (time interval).The electrode loss by corrosion in saliva fluid was Ti/bare > Ti/CaPO 4 > Ti/CaPO 4 -L1 > Ti/CaPO 4 -L2 > Ti-CaPO 4 -L3 due to the adsorption of EO on modified Ti/CaPO 4 surface.

Conclusion
The findings of a recent study demonstrated that the development of a titanium surface for use in dental implant applications is not solely dependent on the type of surface modification used, but also on other aspects that may synergize with the modified materials to increase the implant's stability in the oral environment.The following is a reasonable inference to make considering the current results: 1-Anodization is still a simple electrochemical method used to treat the Ti surface to be active for further coating via the formation of a TiO 2 thick layer.2-Anodized Ti-bare sample has high corrosion resistance, especially after 336 h of immersion in saliva solution where it increases from 844 to 3985 ohms.3-ACP-NPs were used to prepare nano flaks structure from the CaPO 4 coat on the Ti implant.4-The prepared Ti/CaPO 4 sample is considered a good bioactive surface.It increases the corrosion resistance from 460 to 1675 Ω after 240 min and to 6890 Ω after 336 h of immersion in saliva solution.5-Selected three EOs added to saliva solution, Cumin, Thyme, and Coriander, synergized with the CaPO 4 coat on the Ti surface to enhance its stability by increasing the corrosion resistance to new high values equal to 14,523, 7525, and 20,421, respectively, after 336 h. of immersion in saliva solution.
Coriander, Thyme, and Cumin were found to have the most synergistic impact when stacked in that sequence.As a result, the chemical structure of the substance found in the oil might be utilised to explain the order in which the ability to preserve Ti/CaPO4 is ranked.Coriander has a chemical that has a linear structure, and this structure improves the capacity of ions to diffuse through the CaPO4 layer, which in turn raises the corrosion resistance.Despite the cumin compound's bulky structure's ability to restrict diffusion, the compound's resistance to corrosion actually reduced.

Figure 3 .
Figure 3. SEM Image of the amorphous CaPO 4 on the anodized Ti surface.

Figure 4 .
Figure 4. Fitting circuit of the EIS data.

Figure 7 .
Figure 7. Representing of the Nyquist plot of the (a) Ti-bare and (b) Ti/CaPO 4 after soaking in saliva for 336 h.(c) Comparison between the charge transfer resistance at different time intervals.

Figure 8 . 9 Figure 9 .
Figure 8.Comparison between the effect of essential oils concentration on Ti/CaPO 4 and charge transfer resistance after soaking 336 h in saliva solution.

Figure 11 .
Figure 11.Polarization curves of different modified surfaces in saliva solution.
Furthermore, the crystal structure of calcium phosphate consisted of two inequivalent Ca 2+ points.The Ca 2+ is bounded to six equivalent O 2− atoms in the first site.Thus, the Ca-O bond lengths are equal to 2.46 Å.On the other hand, the second site of Ca 2+ atoms is bounded to 10 coordinate geometry to 10 oxide atoms (O 2− ).Hence, the Ca-O bond is ranging 2.25-2.72Å.At the same time, the P-O is represented in short and long bond lengths equal to 1.54 and 1.56 Å, respectively 61,62and 71.1° for attributed to miller indices (100),(200), and (211) respectively59,60.Accordingly, TiO 2 exists in crystal structure and point group of hexagonal and 6/mm, respectively.Furthermore, the anatase titania (TiO 2 ) phase formed via anodization was observed in Fig.1a, which has three peaks at 2θ = 22.9, 38.2, 76.3 for corresponding miller indices of (101), (004), and (211), respectively (JCPDS card no.21-1272).The point group and crystal structure are recommended to be P4 2 /mnm and tetragonal, respectively.As represented in Fig.1b, the calcium triphosphate was characterized by the peaks at 2θ =, 26.02, 31.6, and 32.3 o for corresponding miller indices (002), (211), and (300)61,62.Whereas the space group and crystal structure are recommended to be R3̅ m and Trigonal, respectively.

Table 2 .
Representation of the fitting parameters of Ti-bare electrode for 240 min in simulated saliva solution.

Table 3 .
Representation of Ti/CaPO 4 electrode fitting parameters for 240 min in simulated saliva solution.

Table 4 .
Representation of the fitting parameters of Ti-bare electrode for 336 h in simulated saliva solution.

Table 5 .
Representation of Ti /CaPO 4 electrode fitting parameters for 336 h in simulated saliva solution.

Table 6 .
Representation of the fitting parameters of Ti/CaPO 4 electrode for 240 min in simulated saliva solution in the presence of oil L1.

Table 7 .
Representation of the fitting parameters of Ti/CaPO 4 electrode for 240 min in simulated saliva solution in the presence of oil L2.

Table 8 .
Representation of the fitting parameters of Ti/CaPO 4 electrode for 240 min in simulated saliva solution in the presence of oil L3.

Table 9 .
Representation of the fitting parameters of Ti /CaPO 4 electrode for 336 h in simulated saliva solution in the presence of oil L1.

Table 10 .
Representation of the fitting parameters of Ti/CaPO 4 electrode for 336 h. in simulated saliva solution in the presence of oil L2.

Table 11 .
Representation of the fitting parameters of Ti/CaPO 4 electrode for 336 h in simulated saliva solution in the presence of oil L3.
Electrode E